The ribosome, a large ribonucleoprotein enzyme, is universally responsible for translating messenger RNAs (mRNAs) into the encoded protein products. This process is among one of the most fundamental and highly regulated in all living things. Recent advances in the structural biology of protein synthesis have provided atomic resolution structures of the ribosome as well as lower-resolution snapshots of ribosomal complexes trapped in the process of translation. What is currently lacking from mechanistic models of ribosome function is a description of the kinetics governing transitions from one conformational state of the ribosome to the next. Although difficult, and often impossible, to study precisely using bulk biochemical methods, these conformational dynamics have been shown to be of prime importance in translation. The initiation phase of protein synthesis is the focal point for the translational control of gene expression. As such, the initiation pathway serves as a very effective target for small molecule antibiotics, human viral pathogens, and deregulation of initiation is increasingly causally linked to tumorigenesis. The initiation reaction is an amazingly dynamic process, involving the interaction of numerous translation initiation factors (IFs) with the ribosome in a highly-coordinated and specific series of molecular events. We hypothesize that IFs regulate the initiation pathway by precisely altering the stabilities of dynamically heterogeneous conformational intermediates of the initiation machinery. To address this dynamic conformational heterogeneity, we will use single-molecule fluorescence resonance energy transfer (smFRET). smFRET provides a unique tool for characterizing the conformational dynamics of individual molecules, eliminating the population averaging inherent in ensemble studies and revealing the dynamic heterogeneity of the system. These data will help elucidate the basic mechanism of translation initiation, providing crucial kinetic information that has heretofore remained inaccessible in bulk studies. Specifically, we will use these techniques to (1) investigate the dynamics of initiation factor 2 (IF2) and initiator transfer RNA (tRNAi) that regulate tRNAi selection during initiation, (2) determine how coupling of ribosome and tRNAi conformational dynamics control the fidelity of tRNAi and start codon selection, and (3) establish the currently unknown mechanism through which initiation factor 3 (IF3) acts to proofread the fidelity of the initiation reaction. Our ability to correlate the kinetics of critical conformational changes with fundamental biochemical steps in the initiation pathway will aid the development of a complete mechanistic model for this universal and biomedically relevant biological process.